NANOELECTRIC CHIP FOR KINASE PROFILING

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 激酶是一类在细胞信号网络中起关键作用的酶。研究表明，某些激酶的过度表达可能导致癌症。许多基于激酶抑制剂的药物已经上市或处于癌症治疗的临床开发中。对人类基因组的分析已经产生了一个大的蛋白激酶库（超过500种）。因此，开发新的技术对蛋白激酶抑制剂的快速高通量筛选具有重要意义。在这里，我们提出了一个低成本的，多重的，无标记的，和超灵敏的技术激酶分析使用基于纳米电极阵列（NEAs）的纳米电子芯片的发展。单独寻址的NEAs使用垂直排列的碳纳米管（CNT）镶嵌在SiO2中制造，只有末端暴露，其进一步用适当的肽底物官能化并封闭在微流体通道中。在激酶和ATP的存在下，相应的肽底物通过引入带负电荷的磷酸基团进行磷酸化，导致NEA的AC阻抗发生变化。变化的速率与激酶活性和浓度有关。这 该技术可以潜在地使得能够在约5-10微升溶液中同时检测响应于1-100 pM的激酶混合物的多达100种肽底物。这将大大加快激酶谱用于癌症诊断、治疗监测和药物发现。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kinases are a group of enzymes which serve as crucial nodes in cell signaling network. It has been demonstrated that cancers may be resulted from the over expression of certain kinases. Many drugs based on kinase inhibitors are already on the market or in clinical development for cancer treatment. The analysis of the human genome has yielded a large library of (over 500) protein kinases. It is important to develop new technologies for quick high-throughput screening of protein kinase inhibitors. Here we propose the development of a low-cost, multiplex, label-free, and ultrasensitive technique for kinase profiling using a nanoelectronic chip based on nanoelectrode arrays (NEAs). Individually addressed NEAs are fabricated using vertically aligned carbon nanotubes (CNTs) inlaid in SiO2 with only the very end exposed which are further functionalized with proper peptide substrates and enclosed in a microfluidic channel. In the presence of kinases and ATP, the corresponding peptide substrates undergo phosphorylation with the introduction of a negatively charged phosphate group, resulting in a change in the AC impedance of the NEA. The rate of the change is associated with the kinase activity and concentration. This technique could potentially enable the simultaneous detection of up to 100 peptide substrates responding to the mixture of kinases at 1-100 pM in ~5-10 microliter solutions. This will significantly expedite kinase profiling for cancer diagnosis, treatment monitoring, and drug discovery.